manipulation in other filamentous fungi will
reveal many more compounds with bioactivity
potential.
A recent resurrection of forward genetic
screening approach has managed to identify
mutations in three novel secondary
metabolism-regulating elements in A. nidulans
and A. flavus. Genomes of chemically mutagenized A. nidulans strains suffering from dysregulation of sterigmatocystin synthesis were
sequenced to reveal laeB, sntB, and hamI, presumably acting alongside laeA as regulatory
proteins required for the production of sterigmatocystin. This hypothesis is further supported by their conserved presence in the
genomes of other aflatoxigenic fungi, as well as
a radical decrease in sterigmatocystin production in the A. flavus DlaeB strain (Pfannenstiel
et al. 2017). The deletion of laeB in A. nidulans
led to the discovery of two novel phthalide
compounds (3- and 7-methoxyporriolide) and
two dibenzo[1,4]dioxins (gibellulin C and D)
(Lin et al. 2018), indicating that the deletion of
the new regulatory proteins in other fungi may
yet prove to be a fruitful approach to novel
metabolite discovery.
The response of fungi to stress triggers an
arsenal of regulatory machineries, often with a
profound influence on the secondary metabolome of the fungus (reviewed by Brakhage
2013). This happens through signaling pathways, which often converge at master regulator
nodes. The cell wall integrity (CWI) pathway is
one such responsive signal cascade, formally
ending with a terminal MAP kinase. The deletion of this enzyme often leads to an impaired
growth phenotype, and the inhibition of this
pathway has been shown to lead to decreased
titers or complete abolition of SM production,
e.g., penicillin in A. nidulans or gliotoxin in
A. fumigatus (Valiante 2017 and references
therein).
Other MAPK-regulated SMs include fumonisin B1 in
Fusarium verticillioides, or ACT toxin in Alternaria
alternata (Valiante 2017 and references therein).
Similarly, signals sensed by G-protein-coupled receptors embedded in the fungal cell
membrane activate the cAMP/protein kinase A
pathway, which relays developmental signals
and has been shown to activate a silent SM
cluster in A. fumigatus, revealing a novel compound, fumipyrrole (Macheleidt et al. 2015).
Taken together, these findings suggest that the
genetic manipulation of signaling proteins and
regulators may provide insight not only into
general biology but also cryptic secondary
metabolism in fungi.
Stress signals inducing secondary metabolism are not required to be externally provided.
Activation of the amino acid starvation pathway, particularly the Myb-like transcription
factor BasR, in A. nidulans induced the expression of orsellinic acid gene cluster. Interestingly, activation of BasR also appears to
constitute the fungal response to a biotic challenge presented in co-culture with Streptomyces
rapamycinicus (Fischer et al. 2018).
2. Engineering Native Regulatory Elements in
Fungal Biosynthetic Clusters
The plentiful amount of fungal genomic data
produced and made available in the past decade
has enabled mining for, and prediction of, biosynthetic clusters, often containing a cognate
transcriptional regulatory protein. A seminal
study of Brakhage and coworkers proved that
a silent SM cluster could be awakened by
controlling the expression of its native transcription factor using a promoter induced by
the presence of ethanol (Bergmann et al. 2007).
The A. nidulans aspyridone cluster was thus
activated using the alcA promoter. The use of
different regulatory elements, such as promoter
exchange and transcription factor overexpression, with the purpose of discovery and production of natural products in filamentous
fungi has since become a popular research
strategy (Brakhage and Schroeckh 2011).
Unsurprisingly, genetic engineering of such
basic regulatory elements has led to the discovery of a multitude of secondary metabolites
across various species. A biosynthetic cluster’s
regulatory elements, namely, core enzyme promoter (PeqxS) and transcription factor (eqxR)
270
M. Flak et al.
reveal many more compounds with bioactivity
potential.
A recent resurrection of forward genetic
screening approach has managed to identify
mutations in three novel secondary
metabolism-regulating elements in A. nidulans
and A. flavus. Genomes of chemically mutagenized A. nidulans strains suffering from dysregulation of sterigmatocystin synthesis were
sequenced to reveal laeB, sntB, and hamI, presumably acting alongside laeA as regulatory
proteins required for the production of sterigmatocystin. This hypothesis is further supported by their conserved presence in the
genomes of other aflatoxigenic fungi, as well as
a radical decrease in sterigmatocystin production in the A. flavus DlaeB strain (Pfannenstiel
et al. 2017). The deletion of laeB in A. nidulans
led to the discovery of two novel phthalide
compounds (3- and 7-methoxyporriolide) and
two dibenzo[1,4]dioxins (gibellulin C and D)
(Lin et al. 2018), indicating that the deletion of
the new regulatory proteins in other fungi may
yet prove to be a fruitful approach to novel
metabolite discovery.
The response of fungi to stress triggers an
arsenal of regulatory machineries, often with a
profound influence on the secondary metabolome of the fungus (reviewed by Brakhage
2013). This happens through signaling pathways, which often converge at master regulator
nodes. The cell wall integrity (CWI) pathway is
one such responsive signal cascade, formally
ending with a terminal MAP kinase. The deletion of this enzyme often leads to an impaired
growth phenotype, and the inhibition of this
pathway has been shown to lead to decreased
titers or complete abolition of SM production,
e.g., penicillin in A. nidulans or gliotoxin in
A. fumigatus (Valiante 2017 and references
therein).
Other MAPK-regulated SMs include fumonisin B1 in
Fusarium verticillioides, or ACT toxin in Alternaria
alternata (Valiante 2017 and references therein).
Similarly, signals sensed by G-protein-coupled receptors embedded in the fungal cell
membrane activate the cAMP/protein kinase A
pathway, which relays developmental signals
and has been shown to activate a silent SM
cluster in A. fumigatus, revealing a novel compound, fumipyrrole (Macheleidt et al. 2015).
Taken together, these findings suggest that the
genetic manipulation of signaling proteins and
regulators may provide insight not only into
general biology but also cryptic secondary
metabolism in fungi.
Stress signals inducing secondary metabolism are not required to be externally provided.
Activation of the amino acid starvation pathway, particularly the Myb-like transcription
factor BasR, in A. nidulans induced the expression of orsellinic acid gene cluster. Interestingly, activation of BasR also appears to
constitute the fungal response to a biotic challenge presented in co-culture with Streptomyces
rapamycinicus (Fischer et al. 2018).
2. Engineering Native Regulatory Elements in
Fungal Biosynthetic Clusters
The plentiful amount of fungal genomic data
produced and made available in the past decade
has enabled mining for, and prediction of, biosynthetic clusters, often containing a cognate
transcriptional regulatory protein. A seminal
study of Brakhage and coworkers proved that
a silent SM cluster could be awakened by
controlling the expression of its native transcription factor using a promoter induced by
the presence of ethanol (Bergmann et al. 2007).
The A. nidulans aspyridone cluster was thus
activated using the alcA promoter. The use of
different regulatory elements, such as promoter
exchange and transcription factor overexpression, with the purpose of discovery and production of natural products in filamentous
fungi has since become a popular research
strategy (Brakhage and Schroeckh 2011).
Unsurprisingly, genetic engineering of such
basic regulatory elements has led to the discovery of a multitude of secondary metabolites
across various species. A biosynthetic cluster’s
regulatory elements, namely, core enzyme promoter (PeqxS) and transcription factor (eqxR)
270
M. Flak et al.